Antioxidant and antimicrobial wound dressing materials

ABSTRACT

A wound dressing material comprising a polymeric substrate, a silver salt, and a dyestuff to photostabilize the silver salt. The substrate may comprise collagen and/or oxidized regenerated cellulose complexed to Ag + , and the dyestuff may for example be an aniline or acridine dye.

The present invention relates to antimicrobial wound dressing materials,to processes suitable for the preparation of such materials, and to theuse of such materials in the manufacture of wound dressings.

The antimicrobial effect of silver has been known for centuries.However, the precise mode of action of silver salts in killing microbesis yet to be established. It is known that silver salts bind withparticular avidity to DNA and RNA. Silver salts also bind withparticular strength to a variety of organic molecules such as:carboxylic acids, thiols, phenols, amines, phosphates and halogenatedcompounds. Following binding to proteins, those with enzymic activityare usually deactivated. The oxidative-reductive powers of silver andsilver salts must also be reckoned with.

It is known to use metallic silver as an antimicrobial, whether in theform of thin films, nanoparticles or colloidal silver. Chemicalcompounds of silver are also useful as antimicrobials. For example, thefollowing complex silver salts are favoured for use against sensitiveand resistant bacterial strains:

WO91/11206 describes the use of silver alginate salts in wounddressings. WO87/05517 describes silver salts of hyaluronic acid that maybe used as or in antimicrobial wound dressings. These materials tend notto be stable in the presence of light. The silver undergoesphotochemical reduction to metallic silver, causing a darkening of thematerials over time.

WO02/43743 describes light-stabilized antimicrobial wound dressingmaterials in which silver salts are stabilized by the addition of aphotostabilizer selected from the group consisting of ammonium salts,thiosulfates, metal chlorides and peroxides. Such photostabilizers areof limited effectiveness, and will tend to be extracted from thedressing material by wound fluid.

A need therefore remains for improved antimicrobial dressings containinglight-stabilized silver compounds.

Concentrations of reactive oxygen species such as hydroxyl radicals(.OH), singlet oxygen (¹O₂), hydroperoxyl radicals (.OOH),superoxideradical anions (.O₂ ⁻), and hydrogen peroxide (H₂O₂) can risein damaged tissues, producing a condition known as oxidative stress. Thepresence of a low level of reactive oxygen species may be advantageousin the early stages of wound healing by both attracting and activatingmacrophages which engulf and kill bacteria and release cytokines andgrowth factors. Under mild oxidative stress conditions when hydrogenperoxide levels are slightly raised (around 10⁻⁸ to 10⁻⁴ molar), it hasalso been found that the rate of cell proliferation in fibroblastcultures is stimulated. However, prolonged and more severe oxidativestress may delay healing because it will produce chronic inflammation,divert available energy supply towards antioxidant defence at theexpense of tissue reconstruction, and increase levels of matrixmetalloproteinases which cause tissue breakdown. In more severe cases,elevated levels of reactive oxygen species can give rise to hydrogenperoxide-induced senescence or apoptosis (that is, programmed celldeath) or tissue necrosis (that is, uncontrolled cell death andtherefore permanent tissue damage).

Accordingly, the healing of chronic wounds may be assisted by the use ofantioxidant wound dressings that react specifically with excess reactiveoxygen species such as those listed above and hence reduce the level ofoxidative stress.

U.S. Pat. No. 5,667,501 describes compositions comprising chemicallymodified polymers grafted with chemical groups that confer antioxidantactivity as measured by a diphenylpicrylhydrazyl (DPPH) test and thatalso generate low levels of hydrogen peroxide by reaction with molecularoxygen in the wound bed to stimulate macrophage activity and fibroblastproliferation. The compositions may be used to promote the healing ofchronic wounds. Preferably, the polymer is a polymer bearing hydroxyl,carbonyl or amide functional groups, or a polysaccharide bearinghydroxyl functional groups, said functional groups having been convertedto derivatives that are persistent free radicals or precursors ofpersistent free radicals, that is to say they arefree-radical-scavenging antioxidant groups.

U.S. Pat. No. 5,512,321 describes compositions comprisingpolysaccharides grafted with antioxidants on at least one hydroxyl groupof the polysaccharide. The compositions may be used inter alia topromote the healing of chronic wounds. Preferably, the polysaccharide ishyaluronic acid and the antioxidant group comprises a phenol group.

The above antioxidant wound dressing materials are made by multi-stepchemical reactions to achieve covalent bonding of antioxidant moieties,such as hydroquinones or benzimidazole derivatives, to the polymericsubstrate materials.

A need therefore remains for a more simple and inexpensive route toantioxidant wound dressing materials.

In a first aspect, the present invention provides a wound dressingmaterial comprising a polymeric substrate, a silver salt, and a dyestuffto photostabilize the silver salt.

The wound dressing materials according to the present invention may beprovided in the form of gels, beads, flakes, powder, and preferably inthe form of a film, a fibrous pad, a web, a woven or non-woven fabric, afreeze-dried sponge, a foam or combinations thereof. In certainembodiments, the material is selected from the group consisting of wovenfabrics, knitted fabrics, and nonwoven fabrics, all of which may be madeby conventional methods. In other embodiments, the material may comprise(or consist essentially of) a freeze-dried sponge or a solvent-driedsponge. Methods of making freeze-dried and solvent-dried sponges aredescribed in EP-A-1153622 and EP-A-0838491, the entire contents of whichare incorporated herein by reference.

The wound dressing material is typically in sheet form, for examplehaving an area of from about 1 cm² to about 400 cm², in particular fromabout 2 cm² to about 100 cm². The basis weight of the sheet is typicallyfrom about 100 g/m² to about 5000 g/m², for example from about 400 g/m²to about 2000 g/m².

The polymeric substrate may make up at least 50% by weight of the wounddressing material, for example at least 75% by weight or at least 90% byweight. The substrate polymer is usually not water soluble, but it maybe water swellable.

The polymeric substrate may be bioabsorbable or non-bioabsorbable. Theterm “bioabsorbable polymer” refers to a polymer that is fully degradedand absorbed in vivo in the mammalian body.

Suitable non-bioabsorbable polymers include common textile materialssuch as cellulose, processed cellulose such as viscose, polyimide,polyurethane, and also alginates.

Suitable bioabsorbable polymers include those selected from the groupconsisting of collagens, bioabsorbable cellulose derivatives such asoxidized celluloses, galactomannans such as guar/borate,glycosaminoglycans such as cross-linked hyaluronates,polylactides/polyglycolides, polyhydroxybutyrates, and mixtures thereof.

In certain preferred embodiments the polymeric substrate comprises (andmay consist essentially of) a solid bioabsorbable material selected fromthe group consisting of collagens, chitosans, oxidized celluloses, andmixtures thereof.

Oxidized cellulose is produced by the oxidation of cellulose, forexample with dinitrogen tetroxide. This process converts primary alcoholgroups on the saccharide residues to carboxylic acid group, forminguronic acid residues within the cellulose chain. The oxidation does notproceed with complete selectivity, and as a result hydroxyl groups oncarbons 2 and 3 are occasionally converted to the keto form. Theseketone units introduce an alkali labile link, which at pH7 or higherinitiates the decomposition of the polymer via formation of a lactoneand sugar ring cleavage. As a result, oxidized cellulose isbiodegradable and bioabsorbable under physiological conditions.

The preferred oxidized cellulose for practical applications is oxidizedregenerated cellulose (ORC) prepared by oxidation of a regeneratedcellulose, such as rayon. It has been known for some time that ORC hashaemostatic properties, and that application of ORC fabric can be usedto reduce the extent of post-surgical adhesions in abdominal surgery.

The oxidized regenerated cellulose (ORC) can be obtained by the processdescribed in U.S. Pat. No. 3,122,479, the entire content of which isincorporated herein by reference. This material offers numerousadvantages including the features that it is biocompatible,biodegradable, non-immunogenic and readily commercially available. ORCis available with varying degrees of oxidation and hence rates ofdegradation. The ORC may be used in the form of insoluble fibers,including woven, non-woven and knitted fabrics. In other embodiments,the ORC is in the form of water-soluble low molecular weight fragmentsobtained by alkali hydrolysis of ORC.

In certain embodiments, the oxidized cellulose is in the form ofparticles, such as fiber particles or powder particles, for exampledispersed in a suitable solid or semisolid topical medicament vehicle.In particular, the materials preferably contain ORC fibers, wherein avolume fraction of at least 80% of the fibers have lengths in the rangeof 20 μm to 1000 μm. Such a size distribution can be achieved, forexample, by milling an ORC cloth, followed by sieving the milled powderto remove fibers outside the range. Preferably, the average (mean byvolume) length of the ORC fibers is in the range 250 μm to 450 μm. Theselection of ORC fiber lengths in this range results in easy mixing ofthe ORC and other components and highly homogeneous products.

Preferably, the oxidised cellulose has an average molecular weightgreater than 50,000. Such oxidised cellulose is substantially insolublein wound fluids, but will undergo very gradual breakdown intobioresorbable fragments at physiological pH. Preferably, the oxidizedcellulose is not neutralized. However, the present invention encompassesthe use of partially or completely neutralised materials as described inEP-A-0437095. Especially suitable materials are made if theneutralization is carried out partially or completely with the silversalt of a weak acid, such as silver acetate.

Chitin is a natural biopolymer composed of N-acetyl-D-glucosamine units.Chitin may be extracted from the outer shell of shrimps and crabs inknown fashion. The chitin is then partially deacetylated, for example bytreatment with 5M-15M NaOH, to produce chitosan. Complete deacetylationof the chitin is not a practical possibility, but preferably thechitosan is at least 50% deacetylated, more preferably at least 75%deacetylated. Chitosan has been employed for wound treatment in variousphysical forms, e.g. as a solution/gel; film/membrane; sponge; powder orfiber. Chitosan in the free base form is swellable but not substantiallysoluble in water at near-neutral pH, but soluble in acids due to thepresence of ammonium groups on the chitosan chain. The solubility of thechitosan may be reduced by cross-linking, for example withepichlorhydrin. Typically, the average molecular weight of the chitosanas determined by gel permeation chromatography is from about 105 toabout 106.

The collagen useful as the polymeric substrate on the materialsaccording to the present invention may be any collagen, including Type Ior Type II or Type III collagen, natural fibrous collagen,atelocollagen, partially hydrolysed collagens such as gelatin, andcombinations thereof. Natural fibrous collagen, for example of bovineorigin, is suitable. For example, the collagen prepared from bovine hideis a combination of Type I collagen (85%) and Type III collagen (15%).

In certain embodiments of the present invention, the oxidized celluloseis complexed with collagen and/or chitosan to form structures of thekind described in WO98/00180, WO98/00446 or PCT/GB03/004019, the entirecontents of which are expressly incorporated herein by reference. Forexample, the oxidized cellulose may be in the form of milled ORC fibresthat are dispersed in a freeze-dried collagen or chitosan sponge. Thisprovides for certain therapeutic and synergistic effects arising fromthe complexation with collagen.

In particular embodiments, the polymeric substrate comprises (and mayconsist essentially of) a mixture of: (a) collagen and/or chitosan; and(b) oxidized regenerated cellulose, for example in a dry weight ratiorange of from about 90:10 to about 10:90 of collagen/chitosan:ORC,preferably from about 75:25 to about 25:75, and particularly from about60:40 to about 40:60.

Preferably, the amount of silver (as silver ions and metallic silver) inthe materials according to the present invention is from about 0.01 wt %to about 5 wt. %, more preferably from about 0.1 wt % to about 2 wt. %,and most preferably about 0.1 wt. % to about 1 wt. %, most preferablyabout 0.3 wt. %. Lesser amounts of silver could give insufficientantimicrobial effect. Greater amounts of silver could give rise toantiproliferative effects on wound healing cells.

The silver may be introduced by treating the polymeric substratematerial with a silver salt or compound dissolved or dispersed in wateror an organic solvent such as ethanol, for example as described inWO02/43743. Suitable compounds include silver oxide, silver chromate,silver allantoinate, silver borate, silver glycerolate, silver nitrate,silver acetate, silver chloride, silver sulfate, silver lactate, silverbromide, silver iodide, silver carbonate, silver citrate, silverlaurate, silver deoxycholate, silver salicylate, silver p-aminobenzoate,silver p-aminosalicylate, and mixtures thereof. Preferably, the silveris not present as silver sulfadiazine.

In preferred embodiments, the saver may be complexed to the polymericsubstrate material. The term “complex” refers to an intimate mixture atthe molecular scale, preferably with ionic or covalent bonding betweenthe silver and the polymer. The complex preferably comprises a saltformed between an anionic polymer and Ag⁺. Suitably, the anionic polymeris a polycarboxylate. Suitably, the anionic polymer comprises an anionicpolysaccharide or a polyacrylate. Suitable anionic polysaccharidesinclude alginates, hyaluronates, pectins, carrageenans, xanthan gums,sulfated polysaccharides such as dermatan sulfate or sulfated dextrans,and carboxylated cellulose derivatives such as carboxymethyl cellulosesand oxidized celluloses.

The complex of an anionic polymer and silver can be made by a methodcomprising the step of treating an anionic polymer with a solution of asilver salt. Preferably, the solution is an aqueous solution.Preferably, the anionic polymer is substantially insoluble in water atpH7, and the treatment is therefore carried out on the polymer in thesolid state. For example, the polymer may be in the form of solidfibers, sheet, sponge or fabric. In certain embodiments, the anionicpolymer is a salt and the treatment therefore can be regarded as an ionexchange. In other embodiments, the anionic polymer is at least partlyin free acid form, in which case the silver salt is preferably a salt ofa weak acid, for example silver acetate, whereby the anionic polymer isat least partially neutralised by the silver salt. Similar processes aredescribed in EP-A-0437095, the entire content of which is expresslyincorporated herein by reference.

The neutralization reaction can be carried out in water or alcohol alonebut is preferably carried out in mixtures of water and alcohols. The useof a mixture of water and alcohol provides good solubility for the weakacid salts via the water, and the alcohol prevents the anionic polymerfrom excessively swelling, distorting and weakening during theneutralization. Thus the physical properties of the material areretained. Methanol is the preferred alcohol because many of theabove-mentioned salts have good solubility in this alcohol incombination with water. Preferably, the alcohol to water ratio has arange of about 4:1 to 1:4. If the solution becomes too rich in alcohol,some salts may no longer be soluble particularly if the alcohol is otherthan methanol. If the solution becomes too rich in water, some swellingof the polymer will occur as neutralization takes place and there willbe some loss in physical properties such as in the tensile strength ofthe polymer. Other useful alcohols include, for example, ethyl alcohol,propyl alcohol and isopropyl alcohol.

The use of a mild neutralizing agent such as silver acetate allows forcontrol of the degree of neutralization. Use of stoichiometric andchemically equivalent amounts of neutralizing agent and carboxylic acidon the anionic polymer does not produce a 100% neutralized polymer aswould be produced with strong irreversible reactions with bases such assodium hydroxide, sodium carbonate, sodium bicarbonate and ammoniumhydroxide.

Anionic polymers behave as an ion exchanger and will pull out ofsolution the silver cation of any silver salt that is passed over them.The by-product of this exchange is an acid from the salt and by using asalt of a weak organic acid, a weak acid such as acetic acid is producedwhich does no damage to the polymer. Using salts of strong acids such assodium chloride or sodium sulfate produces hydrochloric acid or sulfuricacid by-products respectively, and these strong acids can cause damagesuch as depolymerization of the polymer

When using silver salts of weak acids, the silver ion is exchanged for aproton on the polymer and part of the salt is converted to weak acid.The mixture of acid and salt in the solution results in a bufferedsolution which maintains a fairly constant pH and controls the degree ofneutralization. An equilibrium reaction is established whereby thesilver ions are bound to the acid portion of the polymer and also to thesalt molecules. This partitioning of the silver ions prevents theneutralization of the polymer from going to completion.

Using a stoichiometric amount of, for example, silver acetate bringsabout a 65-75% degree of neutralization of the carboxylic acid groups onan oxidized cellulose polymer. This control of pH by creating a selfgenerating buffered solution and the use of methanol to control theswelling of the material, leads to a partially neutralized material inwhich the physical properties, e.g. tensile strength and shape of thepolysaccharide, are preserved.

The amount of silver salt used is generally about equal to or up totwice the stoichiometric amount of carboxylic acid content of thepolysaccharide. Alternatively, a second charge of a stoichiometricamount of silver salt can be used if the reaction is recharged withfresh solvent and salt after the first charge reaches a constant pH. Thematerial with elevated pH is then washed to remove the excess silversalt and ions therefrom.

In certain embodiments, at least a portion of the wound dressingmaterial comprises a collagen complexed with silver. This can beachieved by treating a collagen with a solution of a silver salt. Thesilver salt may for example be silver acetate or silver nitrate at aconcentration of about 0.01 molar to about 1 molar. The treatment ispreferably carried out at a pH of from about 5 to about 9. It is thoughtthat the silver complexes primarily to the nitrogen-containing sidechains of the collagen amino acids, in particular to lysine,hydroxylysine, asparagine, glutamine and arginine. The silver could alsobind to the sulfhydryl groups of methionine and cysteine residues, wherepresent, and to carboxyl groups of aspartate and glutamate.

Preferably the amount of silver in the collagen complex is from about0.01 to about 30% by weight based on the weight of the collagen, morepreferably from about 0.1% to about 20%, more preferably from about 2%to about 10% by weight. Preferably, the amount of silver-collagencomplex in the wound dressing material is from about 0.1 to about 10 wt%, more preferably from about 0.1 to about 2 wt. %. In any case, thetotal amount of silver in the wound dressing material is generally asspecified above.

It will be appreciated that the complexes of silver with polysaccharidesdescribed above may be prepared with a relatively high silver content,for example greater than 5 wt. %, and then diluted with furtherpolysaccharide (the same or different) to achieve the desired overallsilver content of from 0.01 wt. % to 5 wt. %, preferably from about 0.2wt. % to about 2 wt. %.

The term “dyestuff” refers to a material that is useful as a colorantfor textile materials, that is to say an organic compound that isstrongly light-absorbing in the visible region 400-700 nm and that bindsin substantially water-tight fashion to textile materials such ascellulose fabrics. The dyestuffs can stabilize the silver salts againstphotochemical decomposition by absorbing light near the surface of thematerial. The dyestuffs also trap photochemically generated freeradicals that could otherwise react with the silver. In this way thedyestuffs can act as photochemical desensitisers.

The dyestuffs may be any suitable, medically acceptable dyestuff thatstabilizes silver salts against photochemical reduction to metallicsilver. Medically acceptable organic desensitisers of the kind used inphotography are suitable. Also suitable are the so-called antioxidantdyestuffs.

In certain embodiments, the antioxidant dyestuff is selected from thegroup consisting of aniline dyes, acridine dyes, thionine dyes,bis-naphthalene dyes, thiazine dyes, azo dyes, anthraquinone dyes, andmixtures thereof. For example, the antioxidant dyestuff may be selectedfrom the group consisting of gentian violet, aniline blue, methyleneblue, crystal violet, acriflavine, 9-aminoacridine, acridine yellow,acridine orange, proflavin, quinacrine, brilliant green, trypan blue,trypan red, malachite green, azacrine, methyl violet, methyl orange,methyl yellow, ethyl violet, acid orange, acid yellow, acid blue, acidred, thioflavin, alphazurine, indigo blue, methylene green, and mixturesthereof.

The dyestuff may be present in the wound dressing material according tothe invention in an amount of from about 0.05% to about 5 wt. %,typically about 0.2 to about 2 wt. % based on the dry weight of thematerial.

The step of dyeing may be carried out either before, concurrently with,or after the step of silver treatment, but preferably it is not carriedout after the treatment with silver in order to avoid leaching of silverinto the dye bath.

It has been found that medically acceptable polymeric substratematerials such as oxidized regenerated cellulose have excellent avidityfor dyes, such as the antioxidant dyes listed above. This enablescontrolled amounts of the dyes to be fixed onto the substrate materialsin a simple and inexpensive dyeing step. The dyes act as photochemicaldesensitizers to stabilize the silver salts against photochemicalreduction. It has further been found that, when antioxidant dyes areused, the resulting dyed materials retain the antioxidant properties ofthe dyestuff, thereby making them excellent candidates for the treatmentof chronic wounds and other wounds characterised by elevated levels ofoxygen free radicals.

The wound dressing materials according to the present invention may alsocomprise up to 20% by weight, preferably less than 10% by weight ofwater. The material may also contain 0-40% by weight, preferably 0-25%by weight of a plasticiser, preferably a polyhydric alcohol such asglycerol. The material may also comprise 0-10% by weight, preferably0-5% by weight of one or more additional therapeutic wound healingagents, such as non-steroidal anti-inflammatory drugs (e.g.acetaminophen), steroids, antibiotics (e.g. penicillins orstreptomycins), antiseptics other than silver (e.g. chlorhexidine), orgrowth factors (e.g. fibroblast growth factor or platelet derived growthfactor). All of the above percentages are on a dry weight basis.

The wound dressing material according to the present invention ispreferably sterile and packaged in a microorganism-impermeablecontainer.

Preferably, the material according to the present invention has a freeradical activity, that is to say an antioxidant activity, of at leastabout 15% in the diphenylpicrylhydrazyl (DPPH) test, measured aspercentage reduction in absorbance at 524 nm after 4 hours of a 0.5% w/vdispersion of the polysaccharide in 10⁻⁴M DPPH, as described furtherhereinbelow in Procedure 1. Preferably the percentage reduction inabsorbance in the DPPH test (after correction for any absorbance by thedye) is at least about 25%, more preferably at least about 50%, and mostpreferably at least about 75%.

Alternatively or additionally, the material according to the presentinvention may exhibit antioxidant activity as measured by its ability toinhibit the oxidation of ABTS (2,2′-azino-di-[3-ethylbenzthiazolinesulphonate]) by a peroxidase.

Preferably, the materials according to the present invention exhibitantimicrobial ability against at least Staphylococcus aureus andPseudomonas aeruginosa, and preferably also against Salmonellacholeraesuis and Candida albicans, as determined by the zone ofclearance test described hereunder as Procedure 2.

Preferably, the material according to the present invention will absorbwater or wound fluid and hence become wet, swell or become a gelatinousmass but will not spontaneously dissolve or disperse therein. That is tosay, it is hydrophilic but has a solubility of preferably less thanabout 1 g/liter in water at 25° C. Low solubility renders such materialsespecially suitable for use as wound dressings to remove reactive oxygenspecies from the wound fluid.

The antioxidant and antimicrobial properties of the materials accordingto the present invention suggest applications in a range of medicalapplications, including the treatment of acute surgical and traumaticwounds, burns, fistulas, venous ulcers, arterial ulcers, pressure sores(otherwise known as decubitus ulcers), diabetic ulcers, ulcers of mixedaetiology, and other chronic or necrotic wounds and inflammatory lesionsand disorders. The materials according to the present invention areprimarily intended for the treatment of both infected wounds andnon-infected wounds (that is to say wounds showing no clinical signs ofinfection).

Accordingly, in a further aspect the present invention provides the useof a material according to the present invention for the preparation ofa medicament for the treatment of a wound. Preferably, the wound is achronic wound and/or an infected wound. More preferably, the chronicwound is selected from the group consisting of ulcers of venous,arterial or mixed aetiology, decubitus ulcers, or diabetic ulcers.

In a related aspect, the present invention provides a method oftreatment of a wound in a mammal comprising applying thereto atherapeutically effective amount of a material according to the presentinvention. Preferably, the wound is a chronic wound.

In a further aspect, the present invention provides a wound dressingcomprising a wound dressing material according to the present invention.

The wound dressing is preferably in sheet form and comprises an activelayer of the material according to the invention. The active layer wouldnormally be the wound contacting layer in use, but in some embodimentsit could be separated from the wound by a liquid-permeable top sheet.Preferably, the area of the active layer is from about 1 cm² to about400 cm², more preferably from about 4 cm² to about 100 cm².

Preferably, the wound dressing further comprises a backing sheetextending over the active layer opposite to the wound facing side of theactive layer. Preferably, the backing sheet is larger than the activelayer such that a marginal region of width 1 mm to 50 mm, preferably 5mm to 20 mm extends around the active layer to form a so-called islanddressing. In such cases, the backing sheet is preferably coated with apressure sensitive medical grade adhesive in at least its marginalregion.

Preferably, the backing sheet is substantially liquid-impermeable. Thebacking sheet is preferably semipermeable. That is to say, the backingsheet is preferably permeable to water vapour, but not permeable toliquid water or wound exudate. Preferably, the backing sheet is alsomicroorganism-impermeable. Suitable continuous conformable backingsheets will preferably have a moisture vapor transmission rate (MVTR) ofthe backing sheet alone of 300 to 5000 g/m²/24 hrs, preferably 500 to2000 g/m²/24 hrs at 37.5° C. at 100% to 10% relative humiditydifference. The backing sheet thickness is preferably in the range of 10to 1000 micrometers, more preferably 100 to 500 micrometers. It has beenfound that such moisture vapor transmission rates allow the wound underthe dressing to heal under moist conditions without causing the skinsurrounding the wound to macerate.

Suitable polymers for forming the backing sheet include polyurethanesand poly alkoxyalkyl acrylates and methacrylates such as those disclosedin GB-A-1280631. Preferably, the backing sheet comprises a continuouslayer of a high density blocked polyurethane foam that is predominantlyclosed-cell. A suitable backing sheet material is the polyurethane filmavailable under the Registered Trade Mark ESTANE 5714F.

The adhesive (where present) layer should be moisture vapor transmittingand/or patterned to allow passage of water vapor therethrough. Theadhesive layer is preferably a continuous moisture vapor transmitting,pressure-sensitive adhesive layer of the type conventionally used forisland-type wound dressings, for example, a pressure sensitive adhesivebased on acrylate ester copolymers, polyvinyl ethyl ether andpolyurethane as described for example in GB-A-1280631. The basis weightof the adhesive layer is preferably 20 to 250 g/m², and more preferably50 to 150 g/m². Polyurethane-based pressure sensitive adhesives arepreferred.

Further layers of a multilayer absorbent article may be built up betweenthe active layer and the protective sheet. For example, these layers maycomprise an absorbent layer between the active layer and the protectivesheet, especially if the dressing is for use on exuding wounds. Theoptional absorbent layer may be any of the layers conventionally usedfor absorbing wound fluids, serum or blood in the wound healing art,including gauzes, nonwoven fabrics, superabsorbents, hydrogels andmixtures thereof. Preferably, the absorbent layer comprises a layer ofabsorbent foam, such as an open celled hydrophilic polyurethane foamprepared in accordance with EP-A-0541391, the entire content of which isexpressly incorporated herein by reference. In other embodiments, theabsorbent layer may be a nonwoven fibrous web, for example a carded webof viscose staple fibers. The basis weight of the absorbent layer may bein the range of 50-500 g/m², such as 100-400 g/m². The uncompressedthickness of the absorbent layer may be in the range of from 0.5 mm to10 mm, such as 1 mm to 4 mm. The free (uncompressed) liquid absorbencymeasured for physiological saline may be in the range of 5 to 30 g/g at25°. Preferably, the absorbent layer or layers are substantiallycoextensive with the active layer.

The wound facing surface of the dressing is preferably protected by aremovable cover sheet. The cover sheet is normally formed from flexiblethermoplastic material. Suitable materials include polyesters andpolyolefins. Preferably, the adhesive-facing surface of the cover sheetis a release surface. That is to say, a surface that is only weaklyadherent to the active layer and the adhesive on the backing sheet toassist peeling of the adhesive layer from the cover sheet. For example,the cover sheet may be formed from a non-adherent plastic such as afluoropolymer, or it may be provided with a release coating such as asilicone or fluoropolymer release coating.

Typically, the wound dressing according to the present invention issterile and packaged in a microorganism-impermeable container.

In a further aspect, the present invention provides a method ofmanufacture of an antioxidant wound dressing material, comprising thesteps of: dyeing a polymeric substrate material with a dye whichstabilizes silver salts against photochemical reduction; and treatingthe substrate material with a silver salt dissolved or dispersed inwater or an organic solvent.

The method according to the present invention may be used to prepare awound dressing material according to the present invention.

The method of the present invention may comprise dyeing a substratematerial in sheet form, for example a woven, nonwoven or knitted fabricor sponge sheet of the substrate material by immersing it in a dye bath,followed by washing to remove unbound dye, treatment with a silver saltas hereinbefore described, and drying. In other embodiments, thesubstrate material may be dyed while it is in fibrous or particulateform, followed by forming the material into a sheet. For example, aslurry of fibers or particles of the substrate material may be treatedwith dye, and then freeze-dried to form a dyed sponge. The silvertreatment may be carried out on the slurry, or on the sponge afterfreeze-drying.

It will be appreciated that any feature or embodiment that is describedherein in relation to any one aspect of the invention may also beapplied to any other aspect of the invention.

Certain specific embodiments of the present invention will now bedescribed further in the following examples.

EXAMPLE 1

An antioxidant and antimicrobial wound dressing material based on acollagen/ORC freeze-dried sponge material is prepared as follows.

The collagen component is prepared from bovine corium as follows. Bovinecorium is split from cow hide, scraped and soaked in sodium hypochloritesolution (0.03% w/v) to inhibit microbial activity pending furtherprocessing. The corium is then washed with water and treated with asolution containing sodium hydroxide (0.2% w/v) and hydrogen peroxide(0.02% w/v) to swell and sterilize the corium at ambient temperature.The corium splits then undergo an alkali treatment step in a solutioncontaining sodium hydroxide, calcium hydroxide and sodium bicarbonate(0.4% w/v, 0.6% w/v and 0.05% w.v, respectively) at pH greater than12.2, ambient temperature, and for a time of 10-14 days, with tumbling,until an amide nitrogen level less than 0.24 mmol/g is reached. Thecorium splits then undergo an acid treatment step with 1% hydrochloricacid at ambient temperature and pH 0.8-1.2. The treatment is continuedwith tumbling until the corium splits have absorbed sufficient acid toreach a pH less than 2.5. The splits are then washed with water untilthe pH value of corium splits reaches 3.0-3.4. The corium splits arethen comminuted with ice in a bowl chopper first with a coarsecomminution and then with a fine comminution setting. The resultingpaste, which is made up in a ratio of 650 g of the corium splits to 100g of water, as ice, is frozen and stored before use in the next stage ofthe process. However, the collagen is not freeze-dried before admixturewith the ORC & other components in the next stage.

The ORC component of the freeze-dried pad is prepared as follows. ASURGICEL cloth (Johnson & Johnson Medical, Arlington) is milled using arotary knife cutter through a screen-plate, maintaining the temperaturebelow 60° C.

Methylene blue, an acidic dye, was incorporated by dissolving anappropriate amount of the dye in 0.05M acetic acid and adding to thecollagen paste with the milled ORC powder to obtain a slurry. Sampleswere made in which the dye was incorporated at the followingconcentrations in the final slurry: 0% (reference example), 1 mg/ml, 0.5mg/ml and 0.1 mg/ml.

Silver is incorporated by dissolving silver acetate in 0.05M acetic acidand adding the solution to the slurry to achieve a final solidsconcentration in the slurry of about 1% w/w. The silver acetate is addedin an amount sufficient to produce a final slurry containing 1 wt. %silver on a total solids basis.

The final slurry was poured into petri dishes to a depth of 3 mm, andplaced onto freezer shelves where the temperature has been preset to−40° C. The freeze-drier programme was then initiated to dry anddehydrothermally cross-link the collagen and ORC to form sponge pads. Oncompletion of the cycle, the vacuum was released, sponge samples werethen packaged, and sterilized by cobalt 60 gamma-irradiation.

EXAMPLE 2

The procedure of Example 1 was followed, but replacing the methyleneblue dye by crystal violet, a basic dye. The crystal violet wasincorporated at the following concentrations in the slurry: 0%(reference example), 1 mg/ml, 0.5 mg/ml and 0.1 mg/ml.

EXAMPLE 3

The procedure of Example 1 was followed, but replacing the methyleneblue dye by flavin 3,6-Diaminoacridine hemisulfate, a basic dye. Theflavin was incorporated at the following concentrations in the slurry:0% (reference example), 1 mg/ml, 0.5 mg/ml and 0.1 mg/ml.

EXAMPLE 4

The procedure of Example 1 was followed, but replacing the methyleneblue dye by flavin 3,6-Diaminoacridine hemisulfate, a basic dye. Theflavin was incorporated at the following concentrations in the slurry:0% (reference example), 1 mg/ml, 0.5 mg/ml and 0.1 mg/ml.

EXAMPLE 5

The procedure of Example 1 was followed, but replacing the methyleneblue dye by a mixture of methylene blue and flavin 3,6-Diaminoacridinehemisulfate, each dye being incorporated in the slurry at aconcentration of 0.5 mg/ml.

EXAMPLE 6

The procedure of Example 1 was followed, but replacing the methyleneblue dye by a mixture of crystal violet and flavin 3,6-Diaminoacridinehemisulfate, each dye being incorporated in the slurry at aconcentration of 0.5 mg/ml.

EXAMPLE 7

The procedure of Example 1 was followed, but replacing the methyleneblue dye by a mixture of crystal violet and methylene blue, each dyebeing incorporated in the slurry at a concentration of 0.5 mg/ml.

The sponges according to the invention obtained in Examples 1 to 7 allshowed stable absorption of the dyes. The sponges could be been soakedin serum at 25° C. for a number of days and remained coloured at alltimes. Depending on concentration of dye added there was an initialrelease of the excess dye and then a gradual release as the spongesbegan to degrade.

Procedure 1

The ability of the wound dressing materials to react with and removeoxygen containing free radicals is assessed by the DPPH test describedin WO94/13333, the entire content of which is expressly incorporatedherein by reference. The test is adapted from that described by Blois M.S. in Nature 181: 1199 (1958), and Banda P. W. et al., in AnalyticalLetters 7: 41 (1974).

Briefly, the wound dressing material under test (2.5 mg; 5 mg; & 25 mgsample sizes) was suspended in 2.5 ml of 0.1M pH 7.0 phosphate buffer. Asolution of diphenylpicrylhydrazyl (DPPH) in methanol (10−4 M) was addedin an amount of 2.5 ml and the mixture was shaken and stored in the darkat 20° C. The samples were assessed by measurement of their lightabsorbance at 524 mm over 6 hours in comparison with a control,particular attention being paid to the figure after 4 hours. Thepercentage reduction of absorbance relative to the control after 4 hoursgives the DPPH test value, with a reproducibility generally of ±5%. Thisvalue may conveniently be expressed in terms of a simple reduction inabsorbance units (AU) relative to the control.

Ascorbic acid, a well-known antioxidant, provides a useful positivecontrol substance for comparative purposes. Freeze-dried sponges ofchitin/chitosan and hydroxyethyl cellulose were used as negativecontrols.

Application of this test resulted in DPPH test values of 80-90% for thepositive control (10⁻⁴M). In contrast, the negative controlschitin/chitosan and hydroxyethyl cellulose exhibited much lower DPPHvalues of less than 15%. The collagen/ORC without any added dye orsilver exhibited some activity in the DPPH test, indicating that ORCitself has some antioxidant properties. The materials according to thepresent invention are expected to exhibit significantly higher activityin the DPPH test than collagen/ORC alone, consistent with antioxidantactivity of the dyes.

Procedure 2

The bactericidal activity of the sponges prepared in Examples 1 to 7 istested on pseudomonas Aeruginosa and staphylococcus Aureus by looking atzone of inhibition.

Six 2 cm×2 cm squares of each sample are cut out in sterile conditions.On day one of the experiment, cultures of both Pseudomonas aeruginosa(ATCC 27853 and various PSI strains) and Staphylococcus aureus (providedby the Dept of Clinical Microbiology and Pathology) are incubatedaerobically at 37° C. for 24 hours on Diagnostic Sensitivity Agar (DSA).After 24 hours test samples are each placed on a DSA plate andimmediately wetted with 0.5 mls of a buffer solution. Three squares ofsample are placed on plates inoculated with Pseudomonas aeruginosa andthree are placed on plates inoculated with Staphylococcus aureus. Theplates are then incubated at 37° C. for 24 hours. The zone of inhibitedgrowth around the sample is then measured using calipers, and the testsample is placed on a new inoculated DSA plate. A swab test is carriedout on the area beneath the sample to determine if the sample isbacteriostatic if not bactericidal by smearing the swab on a DSA plateand incubating it for 24 hours and then examining the growth. Thesamples are transferred onto fresh inoculated plates with the aboveprocedure being carried out every 24 hours for 72 hours as long as thesamples remain intact.

As a negative control, a freeze dried sponge of 45% ORC/55% collagenwithout any silver or dye was tested. A commercially availablesilver-containing antimicrobial dressing (ACTICOAT, registered trademark of Smith & Nephew) and silver nitrate solution (0.5%) were used aspositive controls and zones of inhibition were observed for both overthe test period.

It is found that significant bactericidal effects are observed againstStaphylococcus aureus and Pseudomonas Aeruginosa for the materialsaccording to the invention. The performance of the materials containing1% silver and above is expected to be comparable to that of the ACTICOATdressing.

The above embodiments have been described by way of example only. Manyother embodiments failing within the scope of the accompanying claimswill be apparent to the skilled reader.

1. A wound dressing material comprising a polymeric substrate, a silversalt, and a dyestuff to photostabilize the silver salt. 2-19. (canceled)